Abstract: he seeds from mature fruit of Dendrobiun chrysotoxum Lindl were used as explants to study the suitable medium for aseptic seedlings proliferation and rooting, and the culture condition of aseptic seedlings proliferation was optimized. The results showed that aseptic seedling could be formed directly from seeds, then through its proliferation and rooting to generate complete plants, thus to realize the large-scale production of Dendrobiun chrysotoxum seedling. The suitable culture medium was selected, the optimal medium for aseptic seedling proliferation was MS+0.5 mg/L NAA+2.0 mg/L BA+50 g/L banana puree, proliferation coefficient was 4.7 and they grew well; while the optimal medium for rooting was 1/2MS+0.3 mg/L NAA, the roots of root seedlings were long and thick, the leaves were also thick. Optimization conditions of proliferation: the proliferation coefficient was the highest and plants grew the best while the suitable sugar concentration was 30 g/L, the suitable organic additive was banana puree and the suitable clump planting bud number was 5 each.